RESUMO
Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.
Assuntos
Embrião de Galinha , Modelos Animais de Doenças , Listeria monocytogenes/patogenicidade , Animais , BioensaioRESUMO
Listeria monocytogenes is a bacterial pathogen capable of causing severe infections in humans, often with fatal outcomes. Many different animal models exist to study L. monocytogenes pathogenicity, and we have investigated the chicken embryo as an infection model: What are the benefits and possible drawbacks? We have compared a defined wild-type strain with its isogenic strains lacking well-characterized virulence factors. Our results show that wild-type L. monocytogenes, already at a relatively low infection dose (~5 × 10(2) cfu), caused death of the chicken embryo within 36 h, in contrast to strains lacking the main transcriptional activator of virulence, PrfA, or the cytolysin LLO. Surprisingly, strains lacking the major adhesins InlA and InlB caused similar mortality as the wild-type strain. In conclusion, our results suggest that the chicken embryo is a practical model to study L. monocytogenes infections, especially when analyzing alternative virulence pathways independent of the InlA and InlB adhesins. However, the route of infection might be different from a human infection. The chicken embryo model and other Listeria infection models are discussed.
Assuntos
Embrião de Galinha , Modelos Animais de Doenças , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Animais , Regulação Bacteriana da Expressão Gênica , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5' and 3' untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.
Assuntos
Bactérias/metabolismo , Bactérias/patogenicidade , RNA Bacteriano/metabolismo , Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismoRESUMO
Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5'-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.
Assuntos
Proteínas de Bactérias/genética , Listeria monocytogenes/genética , Fatores de Terminação de Peptídeos/genética , Sequências Reguladoras de Ácido Ribonucleico , Regiões 5' não Traduzidas , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Dados de Sequência Molecular , Temperatura , VirulênciaRESUMO
The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.
Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , RNA Bacteriano/genética , Transcrição Gênica/genética , Animais , Genes Bacterianos/genética , Genoma Bacteriano/genética , Intestinos/microbiologia , Camundongos , Fases de Leitura Aberta/genética , Óperon/genética , RNA Bacteriano/análise , Sequências Reguladoras de Ácido Ribonucleico/genética , Regiões não Traduzidas/genética , Virulência/genéticaRESUMO
The Gram-positive bacterium Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. Expression of five of these factors has previously been shown to be subjected to post-transcriptional regulation as a result of their long 5'-untranslated region (5'-UTR). We have investigated the presence of 5'-UTRs among the other known virulence genes and genes that encode putatively virulence-associated surface proteins. Our results strongly suggest that L. monocytogenes controls many of its virulence genes by a mechanism that involves the 5'-UTR. These findings further emphasize the importance of post-transcriptional control for L. monocytogenes virulence.